Site-Specific Lipidation of a Small-Sized Protein Binder Enhances the Antitumor Activity through Extended Blood Half-Life

Abstract To improve the effectiveness of Factor VIII replacement therapy for Hemophilia A, we sought to develop a PEGylated Factor VIII that would effectively treat bleeding episodes, while reducing the frequency of intravenous injections required for prophylaxis. Previously, we found that the site-specific PEGylation of Factor VIII (PEG-FVIII) preserves full clotting activity, prolongs circulating half-life and extends therapeutic efficacy in a number of bleeding models in hemophilic mice. To further characterize its activity, four naïve Hemophilia A dogs were treated with either PEG-FVIII or unmodified BDD-FVIII in a cross-over study design. All treated dogs showed normalized Whole Blood Clotting Time (WBCT), whole blood Thromboelastograph (TEG) profile, and Cuticle Bleeding Time within 30 min from dosing. Pharmacokinetic analysis of the decay of plasma FVIII activity and antigen levels showed that PEG-FVIII achieved 2-fold longer half-life and reduced clearance and volume of distribution relative to BDD-FVIII. Consistently, PEG-FVIII also demonstrated significantly prolonged efficacy relative to BDD-FVIII by measurement of WBCT and TEG. Both BDD-FVIII and PEG-FVIII were well tolerated in naïve HemA dogs, normal hematology and serum chemistry values were observed following administration. However, two naive dogs that received BDD-FVIII and one naive dog that received PEG-FVIII developed detectable neutralizing antibodies to human FVIII as early as on day 9 post-treatment. In summary, consistent with our previously reported findings in hemophilic mice, in comparison to BDD-FVIII, PEG-FVIII demonstrated superior half-life, full activity in stopping acute bleeding and prolonged efficacy in hemophilia A dogs. Taken together, the results support the use of site-specific PEGylation to create a homogeneous therapeutic for both prophylactic and on-demand treatment of patients with Hemophilia A.

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Phase 1 dose escalation study of MEDI-565, a bispecific T-cell engager that targets human carcinoembryonic antigen (CEA), in patients with advanced gastrointestinal (GI) adenocarcinomas.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.320 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 320-320

Author(s):

Michael J. Pishvaian ◽

Michael Morse ◽

Jennifer T. McDevitt ◽

Song Ren ◽

Gabriel Robbie ◽

...

Keyword(s):

T Cell ◽

Antitumor Activity ◽

Dose Escalation ◽

Half Life ◽

High Titer ◽

Phase 1 ◽

Single Chain ◽

Dose Escalation Study ◽

Human T Cell ◽

Grade 3

320 Background: MEDI-565, a bispecific single-chain antibody, targets human CEA on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex. In murine models, MEDI-565 showed antitumor activity in CEA-expressing tumors (J Immunother 2009;34:341-52). Methods: This phase 1, multicenter, open-label, dose-escalation study enrolled adults with GI adenocarcinomas (including esophageal, gastric, small intestine, colorectal, biliary tract, and pancreatic). MEDI-565 was given intravenously over 3 h on days 1–5 in 28-day cycles, with 4 single-patient (pt) (0.75–20 μg) and 5 standard 3+3 escalation (60 μg–3 mg; 1.5–7.5 mg with dexamethasone [dex]) cohorts. Primary objective was to determine the maximum tolerated dose (MTD); secondary objectives were to evaluate pharmacokinetics (PK), antidrug antibody (ADA), and antitumor activity. Results: Study enrolled39 pts: mean age 59 y; 56% male; 28 (72%) colorectal, 6 (15%) pancreatic, 5 (13%) other. Dose-limiting toxicities (grade ≥ 3 nonhematologic) were seen in 4 pts (2 at 3-mg; 2 at 7.5-mg + dex): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Grade 3 treatment-related adverse events (AEs) seen in 5 pts: diarrhea, CRS, increased alanine aminotransferase, hypertension (all n = 1), and hypoxia (n = 2). Treatment-related serious AEs seen in 6 pts: diarrhea, vomiting, pyrexia, CRS (all n = 1), and hypoxia (n = 2). Five pts discontinued treatment due to AEs: diarrhea, CRS, central nervous system metastases, and hypoxia (n = 2). MEDI-565 exposures increased in approximately dose-proportional manner, with clearance (35–77 L/d) and half-life (2–7 h) typical of drug class. ADA had minor impact; 19 pts (48.7%) had ADAs, 5/39 (12.8%) with high titer, with decreased MEDI-565 concentrations in 2 pts. Plasma inflammatory cytokines were elevated posttreatment in several pts at 1.5- and 3-mg (no dex) dose levels. No objective responses were observed; 11 (28%) pts had stable disease as best response. Conclusions: The MTD of MEDI-565 in pts with GI adenocarcinomas was 5 mg with dex. PK was linear, with fast clearance and short half-life. No objective responses were observed. Clinical trial information: NCT01284231.

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The half-life of RAG-1 protein in precursor B cells is increased in the absence of RAG-2 expression.

Journal of Experimental Medicine ◽

10.1084/jem.183.4.1731 ◽

1996 ◽

Vol 183 (4) ◽

pp. 1731-1737 ◽

Cited By ~ 18

Author(s):

U Grawunder ◽

D G Schatz ◽

T M Leu ◽

A Rolink ◽

F Melchers

Keyword(s):

B Cells ◽

T Cell Receptor ◽

Half Life ◽

Receptor Gene ◽

Cell Receptor ◽

Site Specific ◽

T Cell Receptor Gene ◽

Rag 1 ◽

Precursor B Cells ◽

B And T Lymphocytes

Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that RAG-1 protein turnover in pre-B cells depends on the expression of RAG-2. The apparent half-life of RAG-1 protein is increased when RAG-2 is not expressed in differentiating pre-B cells.

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